Bacterial protease and bacterial amylase compositions

ABSTRACT

ONE OR MORE ODORLESS ZINC COMPOUNDS, PREFERABLY ZINC OXIDE, IS MIXED WITH BACTERIAL PROTEASE IN ORDER TO DECREASE THE TYPICAL PROTEASE ODOR.

United States Patent US. Cl. 195-63 10 Claims ABSTRACT OF THE DISCLOSURE One or more odorless zinc compounds, preferably zinc oxide, 1s mixed with bacterial protease in order to decrease the typical protease odor.

Proteolytic enzymes are well known and bacterial proteases are being used in washing compositions, especially for the purpose of laundering household fabrics and clothng. Enzymes of this type are especially useful for removmg stains caused by blood, chocolate milk, gravy and other proteinaceous food residues. Enzyme compositions containing proteases can be used to make a presoaking bath for articles to be Washed or they can be combined with detergents. Since many washing baths are alkaline, it is desirable that the proteolytic enzyme be sutliciently resistant to alkali so that its activity is not destroyed to the extent that the utility of the enzyme is nullified. Certain bacterial proteases have been developed which retain their activity in a pH range of about 5 to about 10.5 and are otherwise suitable for cleaning uses, either in a soaking or a prewashed product, or in a detergent product.

One of the problems encountered in the preparation of suitable bacterial proteases has been the objectionable odor of the proteases. This problem exists where the bacterial protease is used as such or in combination with bacterial amylase or in conjunction with other additives employed as stabilizing agents or for other purposes. The solution to this problem is complicated by the fact. that enzymes, such as bacterial protease and bacterial amylase are sensitive to various types of chemicals and hence readily subject to loss of activity.

One of the objects of the present invention, therefore, is to provide new and improved bacterial protease compositions in which the characteristic bacterial protease odor is reduced or eliminated. Still another object of the invention is to provide new and improved compositions containing both bacterial protease and bacterial amylase characterized by the fact that the objectionable odor is reduced or eliminated.

Still a further object of the invention is to provide new and improved stabilized compositions containing bacterial protease and/or bacterial amylase which are adapted to be used for the preparation of a presoaking bath for articles to be washed or which can be combined with detergents.

Another object of the invention is to provide a new and improved process for reducing or eliminating the ob jectionable odor of a bacterial protease and/ or a bacterial amylase by a method which does not destroy the protease and/ or amylase activity. Other objects will appear hereinafter.

In accordance with the present invention it has been found that the odor of bacterial proteases, either when used as such or when used in combination with bacterial amylases or stabilizing agents or sequestering agents or other substances, normally employed in the preparation of detergent compositions, can be reduced or eliminated without destroying the enzyme activity by incorporating with thebacterial protease or with a mixture of bacterial protease in combination with one or more of such other materials a small amount of one or more odorless zinc compounds, preferably zinc oxide and/ or zinc hydroxide.

The amount of zinc compound used for the purpose of the invention should be sufficient to reduce the characteristic odor of the bacterial protease. This amount is normally within the range of 0.1% to 3% by weight of a protease composition containing 15,000 to 1,500,000 PV units per gram (PVU/g.). The composition can also contain 0 to 1,500,000 DV units per gram (DVU/g.) of amylase. If the composition contains amylase, a preferred Weight ratio of protease to amylase is 1:1 but this is subject to variation.

The compositions of the invention in which the odor has been inhibited by the addition of at least one odorless zinc compound can also contain stabilizing agents as, for example, calcium acetate monohydrate and inert ingredients or extenders, as, for example, neutral soluble inorganic salts, such as sodium sulfate and/or sodium chloride. In lieu of calcium acetate monohydrate, anhydrous calcium acetate can be used. The calcium acetate serves to stabilize bacterial protease against decomposition, especially in the presence of sequestering agents, such as tripolyphosphate, that are employed in many detergents. Neutral water soluble salts, such as sodium sulfate, are especially useful as carriers and for the purpose of permitting proper proportioning of the bacterial protease when the compositions containing the bacterial protease are used in Wash waters as a preso ak or When they are incorporated in detergent compositions.

The invention will be further illustrated but is not limited by the following examples in which the quantities are by weight unless otherwise indicated.

EXAMPLE I A composition was prepared containing the following ingredients:

Total unitsXlO Ingredients Grams PV DV Bacterial protease Bacterial amylase Calcium acetate monohydra ZHO N 32804 to complete blend Total 100.00 3. 538 2. 979

A composition was prepared by blending the following ingredients in the manner described in Example I:

Total units 10 Ingredients Grams IV DV Bacterial protease.-. 2. 941 0 074 Bacterialamylase. Calcium acetatem oh Total A substantially odor-free product was obtained.

3 4 EXAMPLE III EXAMPLE X A composition was prepared by blending the following ingredients in the manner described in Example I: Total umtsxlm Ingredients Grams PV DV 5 Bacterial protease (PINS-30) 1 7. 30 2. 920 0. 002 gaftierial antylase (A1018) (1 1 1.-. Toawmtsw zlitrirrtifioifiiffljf" :11: Ingredients Grams PV DV N 82304 to complete blend s en it as 02;: a a a BIS g fgf ga 20 n p 12 See the following table.

II O. NazSOr to complete blend- 53.11 DV/gm 1 Bacterial p at as P11530 240 Total 100.00 3.075 1.545 2 l 90,900

Activity of final product:

The product was substantially free from odor. PV/gram 35,380 EXAMPLE IV DV/gram 29,440

EXAMPLE XI 2 parts of enzyme paste containing 490,000 PV units per gram of bacterial protease was dry blended with 5 Totalumtsxlofi parts of calcium acetate monohydrate. 3 mixtures pre- Ingredients Grams P DV pared in this manner were blended, respectively, with 0.1 Baum-m1 protease 1 part zinc oxide, 0.05 part zinc oxide, and 0.025 part zinc l a tenalam ygase (Mgoxide. Each mixture was then finally blended with 15 parts of tripolyphosphate. The odor of each composition ea olto co plete blend was good. These compositions were dissolved in water to Total give a 0.25% solution having pHs of 9.4, 9.3 and 9.25, 12 Same as above Exam 19X respectively which were clear. Each composition was also Activity offinal productf same as above, Example x. assayed and found to contain respectively, 43,920, EXAMPLE XII 44,800 and 40,000 PV units per gram of bacterial protease.

T t1 "ts in EXAMPLE v 3 X Ingredients Grams PV DV A composition was prepared by blendmg 10 parts of a 1 P 5 o 1 wet cake of bacterial protease and bacterial amylase, 25 32222 23%;: 2:920 0:002 parts calcium acetate monohydrate, 0.25 part zinc oxide calcin ng acetate monohydrate and 20.75 parts of sodium sulfate. This composition also f fz2:33:11: was substantially odor-free. This composition contained T H 104,571 PV units per gram of bacterial protease and 40 a 6,333 DV units per gram of bacterial amylase. H am as above, E amp e X- The composition can be further diluted by adding Activity of final product, same as above, Example X. sodium sulfate. EXAMPLE XIII EXAMPLE VI A 't' d b bl d'n 10 parts of Tmlumtsxw compos1 ion was prepare y en 1 g a wet cake of bacterial protease and bacterial amylase Ingredlents Grams PV DV with 25 parts calcium acetate monohydrate, 0.25 part ggge sg sgg lg-pofi of zinc oxide and 20.75 parts of sodium tnpolyphosphate. Calcium-1 acetate ;,;g5 This product assayed 98,000 PV units per gram of bactg gg h terial protease and 6644 DV units per gram of bactenal 2 4 p amylase. It was substantially odorless. Total l 2 Same as above, Exam le X. EXAMPLE VII Activity of final productPsame as above, Example X. The procedure was the same as in Example V except EXAMPLE XIV that the quantity of calcium acetate monohydrate was Total unitsxiu increased to 37.5 parts and the quantity of sodium sulfate was reduced to 8.25 parts. Again a substantially odorless Ingledlents Glam PV DV B t 1 t e Pile-30 1 7. 2.020 0.002 Product was obtamed' 133302221 i ylg e isms) EXAMPLE VH1 z fi stifhifflifiiffli The procedure was the same as in Example VI except Nazso" that the quantity of calcium acetate monohydrate was Total increased to 37.5 parts and the quantity of sodium tn- Same as above Example polyphosphate was decreased to 8.25 parts. Agaln the Activity of final product, same as above, Example X. product was substantially odorless. 55 EXAMPLE XV EXAMPLE IX Total unitsXl0 The products prepared as in Examples V, VI, VII and Ingredients Grams Pv VIII were each dissolved in water at room temperature B t 1 t P115-301 at concentrations of 0.25% by weight giving solut1ons ZZ ZEZ 2 3 7 228 EA108)2 having pHs of 7.4, 7.2, 7.5 and 7.4, respectively. After g fig gg g monohydmtsfl 24 hours, the percent PV retention was determmed and sodium ifgfg gg' 'ggg gg 1,5 5.1: found to be 86.5, 94.8, 83.2 and 91, respectively. Total In the following examples the blending procedure was the same as in Example I. 12 See the following table:

E M PV/Gm. DV/Gm. XA PLE XXII I a 1 Bacterial protease (PMS-30) 400, 000 250 w X10 2 Bacterial amylase (A108)..... 19, 100 90, 900 Ingredients Grams PV DV 5 Bacterial protease (P115-30) 1 7 30 2 920 0 002 Activity of final product: Bacterial amylase (A108) 32. 40 0: 618 21942 PV/ r 53,070 g alglg gcetate monohydrata. 20.00 DV/gram 44,160 Trl ol hcblittb'hlbli'fiifio- EXAMPLE XVI 10 Total 100.00 3, 53s 2. 944

Total units l0 l: garite Esta-130378, Egample X. b E 0 1v 0 111a r0 uct same as a ove, xam Ie X. Ingredients Grams PV DV y p I p B l 0 l p asmas:253 .3}--. EXAMPLE XXIII a cium ace a emono y ate Zinc oxide Total unltsxlo Sodium sulfite to complete blend Ingredients Grams PV DV Total 000 307 4-416 gacgerlal protease l 1 2 Same as above, Example XV. ac amy ase Activity of final product, same as above, Example XV. 23 g iggfffii fft iffi EXAMPLE XVII Sodium sulfite l Tripolyphcsphate to complete blend.

Total unitsXlO Total 100.00 3. 538 2. 944 Ingredients Grams PV DV 1 2 Same as above, Example X. Bacterial protease (P115-30) 10. 950 4. 380 0. 003 Activity of final p e a bov Example Bacterial amylase (A108) 48. 560 0. 927 4. 413 25 Calcium acetate monohydrate Zinc hydroxide EXAMPLE XXIV Sodium sulfate to complete blend T t l 4! Total u o a umtsXlO In edients G V D 1 2 Same as above, Example XV. 3 gr rams P V Activity of final product, same as above, Example XV- 0 Bacterial protease (P115-30) 1 7. 2.920 0.002 EXAMPLE XVIII lziflcgiglagmylase (A108) 33. 0.618 2. 942 Total unitsxloa Tripolyphosphate to complete blend- 60.05

Ingredients Grams V 00 3 3 4 l 2 Same as above Example X. Bacterial protease (Put-sop 10. 950 4.380 0 00a I Bacterial amylase (Amwanu 48. 560 0. 927 4 413 Activity of final product, same as above, Example X. galciumtagctate monohydrate- 000 1116 3GB 8. e Sodium sulfate to complete blend EXAMPLE XXV Total 100. 000 5. 307 4. 416

Total units x10 12 Same as above, Example XV. Activity of final product, same as above, Example XV. Ingfedlents Grams PV DV EXAMPLE XIX gactegal protease (gig-2 0) l 35.30 2.920 0.002 Total unitsX10 Zi rfi: 40 M18 942 S dill lfit Ingredients Grams PV DV T ipofir rfll ospiiate to comp e e lend- Bacterial protease (Pll530) Total-- Bacterial amylase (Al08) 100 00 3 538 2 944 Calcium acetate monohydra 1 2 Same as above, Example X. Zinc phosphate Activity of final product, same as above, Example X. Sodium sulfate to complete blen Total I 2 Same as above, Example XV. EXAMPLE XXVI Activity of final product, same as above, Example XV. T t l units 10 EXAMPLE XX Ingredients Grams PV DV E? umtsxlofi Bacterial Protease (P1l5-30) 1 In redients Grams w w gg g gg ggglz g ggggfgg Bacterial rotease (P1l5-30) 10. 950 4.380 0. 00s g alcmrial gmylase g n 560 0. 927 Sodium sulfite to complete blend a cium acetate mono y Zinc. carbonate Total 100. 00 3. 538 2. 944 Sodium sulfate to complete is f f g b E 1 X Total 0 W1 y 0 ma pro uc same as a ovc, xamp e l 1 Same as above, Example XV. v Activity of final product, same as above, Example X EXAMPLE XXVH EXAMPLE XXI T t l t X10 Total umtsXlG o a um s Ingredients Grams PV DV Ingredients Grams PV DV Bacterial protease (Filo-30) 1 Bacterial protease (Pl15-30) 7.30 2.920 0. 002 Bacterlal amylase (A108) 2 32. 40 2. 942 Calcium acetate monohydrate Bacterial amylase (A108) Calcium acetate monohydrat Zinc oxide Sodium sulfit Sodium sulfate to complete blend Total Zinc oxide Disodlnm salt of ethylene diamlne tetra acetic Thiourea to complete blend.

TotaL.

l 2 Same as above, Example X.

Activity of final product, same as above, Example X.

1 2 Same as above, Example X. Activity of final product, same as above, Example X.

EXAMPLE XXVIII EXAMPLE XXXIV Total units l Total units 10 Ingredients Grams PV DV Ingredients Grams PV DV Bacterial protease (Filo-30) 1 32. 40 12.960 0.008 5 Bacterial protease (P11530) Calcium acetate monohydratm. .00 Zinc oxi Zinc oxide Sodium sulfite Sodium sulfate to complete blend Tripolyphosphate to complete blend T0t81 100.00 12.960 0.008 Total.--:: 100.000 12. 050 0 008 I See the following table: 1 Same as above, Example XXVIII.

Activity of final product, same as above, Example XXVIII. PV/gm. DV/gm Bacterial protease (1 115-30) 400,000 240 EXAMPLE XXXV Activity of final product:

Pll'r/ gram.'. r;-. 7 129, 600 Total umtsxwd D Ingredients Grams PV DV Bacterial protease (PUB-30) 32. 400 12.960 0. 008 Calcium acetate monohydrate 20.0 EXAMPLE XXIX Zinc oxide 0. 250

Sodium sulfite to complete blend. Total units 10 Total 100. 000 12. 960 0.008 Ingredients Grams PV DV 1 Same as above, Example XXVIII. gacteri'ia protease (P115-30) 1 32.400 12.960 0 008 Activity of final product, same as above, Example XXVIII.

inc ox e 0 Sodium sulfate to complete Total 12.960 0. 008

EXAMPLE XXXVI 1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII. Total units 10 Ingredients Grams PV EXAMPLE XXX (laftfrlal prtittzase (P1lh5gO) t acumaceaemono rae Total umtsXl0 Zinc Oxide" y Ingredients Grams PV DV Bacterial protease (Pl15-30) 32.400 12.960 0.008 12 960 0 008 Calcium acetate monohydrate. 20. 000 L 3 5 3 23 i m fi r w ibo v m XX E sodium sulfatih-aix-lb-li-m-end c m y of na pro uc same as above, xample XXVIII.

Total 100.000 12 960 0. 008

1 Same as above, Example XXVIII. EXAMPLE XXXVII Activity of final product, same as above, Example XXVIII. 40

Total units 10 Ingredients Grams PV DV EXAMPLE XXXI Bacterial protease (Pll5-30) 32. 400 12. 960 0. 008 Total unitsxlo Calcium acetate monohydratc. .000

Zinc oxide Ingredients Grams PV DV Thiourea to complete blend Bacterial protease (1115-30) 32. 400 12. 960 0.008 Total 100.000 12. 960 0.008 Calcium acetate monohydra 20.000 Zinc oxide 0.250 l Same as above, Example XXVIII. Tripolyphosphate to comple 47. 350 Activity of final product, same as above, Example XXVIII.

Total 100.000 12.960 0.008 1 S b E 1 XXVIII ame as a ove, xamp e Activity of final product, same as above, Example XXVIII. EXAMPLE XXXVHI Total unitsX10 EXAMPLE XXXI]: Ingredients Grams PV DV Bacterial protease (P115430) 32. 400 12.960 0.008 i fi lt (zlalciuniidacetate monohydrate .000 inc ox e Ingredlems Grams PV Dv Disotiiium salt of ethylene diarnine tetra- Bacterial protease (P1l5-30) 32. 400 12. 960 0.008 W Calcium acetate monohydrate 0.000 Sodmm Sulfate to complete blend Zinc oxide T fl Sodium sulfite a Tr1polyphosphate to complete blen as above Example XXVHL Total 100. 000 12 960 008 Activity of final product, same as above, Example XXVIII.

1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII.

EXAMPLE XXXIX EXAMPLE XXXIII Total unitsXlO Total units 10 Ingredients Grams PV DV Ingredients Grams PV DV Bacterial protease (P1l530) Calcium acetate monohydrate Bacterial protease (P11530) 32. 400 12. 960 0.008 IP 1 q l 0.250 Dlsodlum salt of ethylene diamine tetra- Total 100. 000 0. 008

acetic acid to complete blend Total 1 Same as above, Example XXVIII.

Activity of final product, same as above, Example XXVIII.

1 Same as above, Example XXV lII. Activity of final product, samc as above, Example XXV lIl.

EXAMPLE XL EXAMPLE XLVII Total unitsXlO Total unitsXlO Ingredients Grams PV DV Ingredients Grams PV DV Bacterial protease (P115-30)1 32. 400 12.960 0, cos Bacterial protease 530) l 32. 400 12. 960 0. 008 Calcium acetate monohydrate 20.000 0916mm acetate monohydrflte 20. 000 Zinc oxide o. 250 Zinc carbonate Thiourea I 23. 675 dlum sulaftc to complete blend Disodlum salt of ethylene diamine tetraacetic acid to complete blend 23.675 Total 100.000 12.960 0. 008

Total 100.000 12. 960 o, 003 1 Same as above, Example XVIII.

Activity of final product, same as above, Example XVIII.

1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII.

EXAMPLE XLI EXAMPLE XLVIII Total units l0 15 Total units 10 Ingredients Grams Ingredients Grams ZE V DV Bacterial protease (Filo-30) 1 32. 400 Bacterial protease (PUG-30) 1 32. 400 12.960 0. 00 Calcium acetate monohydrate 20. 000 Calcium acetate monohydrate 20. 000 Zinc hydroxide I 0. 250 Zinc citrate Sodium sulfate to complete blend 47 350 Sodium sulfate to complete blend Total 100. 000 12.960 0.008 Total 100.000 12. 960 0.00

1 Same as above, Example XXVIII. 1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII.

EXAMPLE XLII Total ImitSXlO" EXAMPLE XLIX Ingredients Total unitsXlO Bacterial protease (P1l5-30) 1 Calcium acetate monohydrate Ingredients Zinc borate Sodium sulfate to complete blend Bacterial protease (PUG-) 1 Calcium acetate mollohydratc. Total 100. 000 12. 960 0. 00s Zmc pyrophosphate Sodlum sulfate to complete blend 1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII. Total EXAMPLE XLIII ASettmet as above, Example XXVIII.

c 1 Total unitsxma lvl y 0 ma product, same as above, Example XXVIII.

Ingredients Bacterial protease (P115-30) L EXAMPLE L galciumfiacetate monohydrate inc su e T t 1 10 Sodium sulfate to complete blen I d E m n e ients G V Total 100.000 12.960 0. cos B gt mm P DV acterlal protease (P115-30) 1 32. 400 12.960 0.008 1 s as bove, Example XXVIII. Calcium acetate monoliydrate- 20.000 Activity of final product, same as above, Example XXVIII. Zinc propionate EXAMPLE XLIV Sodium sulfate to complete blend I- Total unitsxm, ota 100.000 12.960 0. 00s

. 1 Same as above Example XXV III. Ingredlents Grams PV DV Activity of finalproduct, same as above, Example XXVIII. Bacterial protease (P115-30) 32. 400 12. 960 0 008 Calcium acetate monohydrate 20.000 Zinc sulfate 0. 250 Sodium sulfate to complete blend. 47. 350 EXAMPLE LI Total 100. 000 12. 960 0. 008

Total unitsXlO 1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII. Ingredients EXAMPLE XLV Bacterial protease (P1l5-30) Bacterial amylase (A108) Total nl X1 Calcium acetate monohydrate M Zinc citrate. Ing edients Grams PV 1)V Sodium sulfate to complete blend Bacterial protease (1 115-30) l m galciumtagetate monohydrate- 1 000 307 416 809 a e l 2 Same as above Example XV. Sodlllm sulfate to complete blend m Activity of final product, same as above, Example XV.

Total 100.000 12. 960 0. 008

1 Same as above, Example XXVIII. Activity of final product, same as above, Example XXVIII. r EXAMPLE LII EXAMPLE XLVI Total unitsXlll Total l1nits 10 Ingredients Grams PV DV Ingredients Grams PV DV Bacterial protease (Pll530) Bacterial protease (P1l5-30) 1 32. 400 12. 960 0. 008 Bacterial amylase (A108) Calcium acetate monohydrate 20. 000 Calcium acetate monohydrate- Zinc phosphate I. Zinc pyrophosphate Sodium sulfate to complete blend Sodium Sulfate to complete blend Total. 100.000 12. 960 0.008 Total 100.000 5. 307 4. 416

l Same as above, Example XXVIII. 12 Same as above, Example XV.

A y o fi a p Same as above, Example V Activity of final product, same as above, Ex ample XV.

EXAMPLE LIII Total unitsXlO Ingredients Grams PV Bacterial protease (P1l530) Bacterial amylase x108). Calcium acetate monohydrat Zinc propionate Sodium sulfate to complete blend Total 1 9 Same as above, Example XV. Activity of final product, same as above, Example XV.

EXAMPLE LIV A commercial dry solid enzyme product known as Alkalase which assayed 123,100 PV/gram was mixed in proportions of parts of said product to 0.1 part of zinc oxide by blending the components and hand grinding the mixture to prepare a uniform blend. The addition of the zinc oxide gave a marked odor reduction without destroying the activity.

- EXAMPLE LV The procedure was the same as in Example IJIV except that a commercial product called Maxatase was used instead of Alkalase. This product had a labeled potency of 300,000 Delft units/ gram (equivalent to 103,000 PV/ gram). The addition of the zinc oxide gave a marked odor reduction without destroying the activity.

EXAMPLE LVI The procedure was the same as in Example DIV except that 2 parts of calcium acetate monohydrate was also added. Again a marked odor reduction was obtained. A similar result was obtained when anhydrous calcium chloride was used instead of the calcium acetate.

EXAMPLE LVII EXAMPLE LVIII Total unitsXlO Ingredients Grams Bacterial amylase (TX 8-80) Calcium acetate monohydrate Zinc oxide Sodium sulfate to complete blen Total...'..--.----....-.

1 Bacterial amylase (TX 8-80) PV/gram DV/gram EXAMPLE LIX Total units 10 Grams Ingredients Bacterial protease (P1l5 30) Calcium chloride anhydrous Zinc oxide N azS O4 to complete blend...

Total 1 Lot No. P1153 PV/g'i'arn 400,000 nv ln-am 240 EXAMPLE LX 1 1 Same as above, Example XV.

It has also been found that calcium acetate monohydrate alone in a blend of 32.40 parts of bacterial protease (P-30), 20 parts of calcium acetate monohydrate and 47.60 parts sodium sulfate inhibits odor. Similarly, 20 parts of anhydrous calcium chloride substituted for the 20 parts calcium acetate monohydrate in the same composition inhibits odor. In another composition containing 7.30 parts bacterial protease (P115-30), 32.40 parts bacterial amylase (A108) and 40.30 parts sodium sulfate, the addition of either 20 parts calcium acetate monohydrate or 20 parts anhydrous calcium chloride inhibited odor.

It will be understood that the compositions described in the examples can be standardized to various levels of PV and DV activity by adding neutral soluble salts, such as sodium sulfate and/or sodium chloride, or by adding additional amounts of calcium acetate monohydrate or by adding sodium tripolyphosphate or sodium sulfite or thiourea or a chelating agent.

Examples of chelating agents which can be used are the disodium salt of ethylene diamine tetraacetic acid, the tetrasodium salt of ethylene diamine tetraacetic acid, the sodium salts of N-(hydroxyethyl)ethylene diamine tetraacetic acid, the sodium salts of diethylene triamine pentaacetic acid, and the sodium salts of nitrylotriacetic acid.

In general, good results are obtained by the use of standardized compositions containing 15,000 to 150,000 PV units per gram and 0 to 150,000 DV units per gram.

One half of one percent (.5%) by weight of the prodnet of Example I when added to a standard laundry detergent will give excellent results in wash waters to which the deteregnt enzyme formulation is added at a concentration of 0.15% and fabrics are washed with such waters at standard conditions (wash cycle of 10 minutes at F. (48.9 C.) and a rinse cycle of 5 minutes at 100 F. (37.7 C.), both at 100 agitator cycles per minute.

In a series of standard wash tests, 0.75% of the enzyme composition of Example XXVIII was mixed with a commercial detergent (Axion base) and used in washing test cloths cut into swatches 6" x 3". The concentration of detergent in the solution Was 0.1%. The washing tests were conducted at a temperature of either 120 F. or F. for 10 minutes followed by a rinse cycle of 5 minutes at 100 F. pH values of the Wash solutions were determined at the end of the wash cycle. Reflectance was determined before and after washing using a Hunter D-25 color difference reflectometer. In one series of tests a standard soiled cloth (EMPA 112) was used and in another series of tests another type of standard soiled cloth (EMPA 116) was used. In a wash test at 120 F. using EMPA 116 cloth the pH at the end of the Wash cycle was 8.75 and the difference in reflectance 39.7. In another wash test at 120 F. using EMPA 112 cloth, the pH at the end of the wash cycle was 8.7 and the difference in reflectance was 24.4. In a third test using a wash cycle of 140 F. with EMPA 112 cloth the pH at the end of the wash cycle was 8.85 and the difference in reflectance was 32.8. In another test which was identical with the last mentioned test except that one gram per liter of wash water of sodium tripolyphosphate was added with the dctergent, the pH was 9.1 and the difference in reflectance was 34.5. Inall cases the results with the compositions of the present invention were better than those obtained with a commercial enzyme and in the case where addi- 13 tional sodium tripolyphosphate was added the difierence in reflectance was 34.5 against 31.5 for a commercial enzyme. All of these tests were conducted on Milwaukee, Wisconsin, city water having a hardness of 135 ppm. as calcium carbonate.

Typical laundry detergent, for example, will contain non-ionic surfactant (e.g., Triton 101), 60% sodium tripolyphosphate, about 20% sodium sulfate, and minor amounts of other additives, such as carboxymethyl cellulose, optical brighteners, and the like.

The invention makes it possible to prepare compositions which will retain their activity over relatively long 'periods of time and which can be stored, handled and incorporated with detergents or used as a presoak prodnet in a very simple manner.

The invention is especially valuable in providing a method for reducing or eliminating bacterial enzyme odors without destroying the enzyme activity. The compositions of the invention do not sufier, therefore, from one of the principal objections inherent in the use of bacterial enzymes, such as bacterial proteases and bacterial amylases.

The invention is hereby claimed as follows:

1. A dry solid composition comprising an enzyme having a characteristic odor from the group consisting of bacterial amylase, bacterial protease and both bacterial amylase and bacterial protease intimately blended with an odorless inorganic zinc compound in an amount suflicient to decrease said odor.

2. A composition as claimed in claim 1 in which the quantity of said compound is within the range of 0.1% to 3 by weight of said composition.

3. A composition as claimed in claim 1 in which the activity of said protease in said composition is within the range of 15,000 to 1,500,000 PV units per gram, and the quantity of said compound is within the range of 0.1% to 3 by weight of said composition.

4. A composition as claimed in claim 3 in which said compound is zinc oxide.

5. A composition as claimed in claim 1 which comprises both bacterial amylase and bacterial protease.

6. A composition as claimed in claim 1 which also comprises an odorless inorganic water soluble calcium compound.

7. A composition as claimed in claim 1 which also comprises a neutral water soluble in organic salt.

8. A dry solid composition comprising 15,000 to 1,500,000 PV units per gram of bacterial protease, 0 to 1,500,000 DV units per gram of bacterial amylase, intimately blended with zinc oxide in suflicient amount to reduce the odor attributable to said bacterial protease and said bacterial amylase.

9. A composition as claimed in claim 8 in which the quantity of zinc oxide is within the range of 0.1% to 3% by weight of the composition.

10. A composition as claimed in claim 9 which also comprises sodium tripolyphosphate.

References Cited UNITED STATES PATENTS 3,164,523 1/1965 Fox et al. -63 X 3,284,316 11/1966 Cayle 195-63 3,451,935 6/1969 Roald et al. 195-63 X 3,524,798 8/1970 Lloyd eta l 195-31 X LIONEL M. SHAPIRO', Primary Examiner D. M. NAFF, Assistant Examiner US. Cl. X.R. 195-68 

